Telomerase is a unique reverse transcriptase that protects genome stability by adding simple- sequence repeats to chromosome termini. Some terminal telomeric repeats are eroded with each cell division due to incomplete DNA-templated DNA polymerase synthesis. By copying its integral RNA template, telomerase can extend a chromosome end to compensate for proliferation-linked repeat loss. Human pluripotent stem cells have active telomerase and therefore long-term renewal capacity, but most human somatic cells lack telomerase function and therefore have limited renewal. Patients with human telomerase deficiencies that cause bone marrow failure, aplastic anemia and pulmonary fibrosis have accelerated telomere shortening causative for tissue failures. Opposite this effect is the telomerase reactivation that underlies the proliferative immortality of almost all human cancers. The long-term objective of research funded by this RO1 is to understand the biochemical and cellular basis of human telomerase ribonucleoprotein (RNP) biogenesis, catalytic activation and regulation in normal cells and disease, and to exploit this understanding for the improvement of human health. Knowledge of human telomerase function and regulation has direct relevance for improvements of human health, for example in designing improved hematopoietic stem cell transplantation for bone marrow failure patients. It will also enable screens for telomerase inhibitors that could be highly effective anti-cancer agents. Studies in mouse models are unable to substitute for studies using human cells due to differences in telomerase structure, telomeric chromatin and telomere length regulation in rodents versus primates. Research in the next funding period will provide fundamental gains in knowledge about how the cellular human telomerase holoenzyme engages telomeres. All three Aims expand results and experimental systems established in the current funding period. Aim 1 studies define the roles and regulations of telomerase-telomere association through the telomere protein TPP1. Embryonic stem cells are the only known human cells that physiologically maintain telomere length homeostasis, so in collaboration with the Hockemeyer lab they will be used for genetic approaches to understand telomerase recruitment and catalytic activation by TPP1. Aim 2 studies use an innovative, redirected telomerase holoenzyme assembly pathway to resolve the functions of H/ACA and Cajal body proteins in telomere elongation versus initial telomerase RNP biogenesis. Aim 3 studies exploit approaches including in vivo protein-RNA crosslinking and in vitro reconstitution to define the biochemical mechanisms of individual steps of telomerase regulation.